In mammals, the vascular endothelial growth factor (VEGF) family consists of five members, VEGF-A, -B, -C, - D and placenta growth factor (PlGF), synthesized by various cell types, including cardiomyocytes. VEGF-B is relatively understudied and yet is emerging as a major pro-survival factor. Similar to PlGF, it binds selectively the receptor VEGFR-1. We have recently found that VEGRF-1 is expressed in cardiomyocytes and provided the first evidence that VEGF-B167 gene transfer reduces myocyte apoptosis and angiotensin II-induced oxidative stress and markedly attenuates cardiac remodeling and functional derangement in dogs with pacing- induced dilated cardiomyopathy. On the other hand, VEGF-A gene delivery did not yield similar beneficial effects, in vivo. The cytoprotective/antiapoptotic and minimally angiogenic effects of VEGF167 render this factor an appealing candidate for gene therapy of non-ischemic dilated cardiomyopathy, which is not caused by coronary artery disease, hence would not benefit from angiogenesis. The overall goal of the present project is to test the hypothesis that VEGFR-1 is the main mediator of VEGF-induced cytoprotection in hearts with non-ischemic dilated cardiomyopathy. Cardiac gene transfer of VEGFR-1 ligands will be performed via adeno-associated vector-9 (AAV9) in chronically instrumented dogs with pacing-induced dilated cardiomyopathy. Parallel experiments will be conducted in vitro to explore molecular mechanisms and to optimize transgene constructs. Aim 1 is to determine the pathophysiological and cellular mechanisms underlying the different effects of VEGFR-1 versus VEGFR-2 stimulation in dilated cardiomyopathy. We will first identify the most cardioprotective VEGFR-1 ligand and then compare it to VEGF-E, a selective VEGFR-2 ligand, and to VEGF-A, a dual ligand, in paced hearts. The molecular basis of VEGFR-1 and VEGFR-2- mediated myocardial protection will be studied in cultured cardiomyocytes. Aim 2 is to determine the optimal timing for VEGF-B-induced cardioprotection during the course of pacing-induced dilated cardiomyopathy. The most cardioprotective VEGFR-1 ligand identified in specific aim 1 will be delivered as an AAV-carried transgene to the heart at different time points during the pacing protocol. Aim 3 is to test the cardioprotective effects of VEGF-B transgenes inducible by endogenous stimuli specifically occurring in the failing heart. An ideal strategy for future clinical applications would be to deliver therapeutic transgenes expressed only in the presence of molecular alterations relevant to the disease. Toward this goal, we will develop the following innovative strategies to achieve VEGF-B transgene expression induced by endogenous stimuli occurring in the failing heart: a) construction of artificial promoters comprising regulatory elements sensitive to oxidative stress; b) utilization of the natural promoter of the atrial natriuretic peptide; c) inclusion, in the 3' UTR region of the transgene, of target sequences for microRNAs that are expressed in the healthy heart, but downregulated in the failing heart. PUBLIC HEALTH RELEVANCE: This project will explore the role of growth factors, named vascular endothelial growth factors (VEGF), in the protection of cardiac cells under a pathological condition known as dilated cardiomyopathy. This cardiac disease is less frequent that others due to coronary occlusion or dysfunction, however it is very malignant, difficult to treat with the available pharmacological agents, and is responsible for more than 50% of cardiac transplants in the US. The administration of VEGFs can be a valid alternative to the current therapies for dilated cardiomyopathy, but, before this new strategy can be implemented, it will be necessary to understand in detail the function and beneficial of these factors as well as their harmful effects. We will therefore utilize a clinically relevant dog model of dilated cardiomyopathy and will test various members of the VEGF family that activate different receptors. Our hypothesis is that those VEGF members that activate the so called VEGF receptor-1 are the ones more likely to display relevant beneficial effects and to be considered for an innovative therapy of dilated cardiomyopathy.